Pathogenicity of Beauveria Bassiana (Deuteromycota

Home , Entomopathogenic fungus, Hyphomycetes, Ophiocordyceps, Ophiocordycipitaceae

Revista Colombiana de Entomología 2019, 45 (2): e8606 • https://doi.org/10.25100/socolen.v45i2.8606

Sección Control / Control Notas científicas / Scientific notes Pathogenicity of Beauveria bassiana (Deuteromycota: Hyphomycetes) to the red cashew beetle Crimissa cruralis (Coleoptera: Chrysomelidae: Galerucinae: Blepharida- group) in laboratory conditions Patogenicidad de Beauveria bassiana (Deuteromycota: Hyphomycetes) al escarabajo rojo anacardo (Coleoptera: Chrysomelidae: Galerucinae: grupo Blepharida) en condiciones de laboratorio SUYANNE ARAÚJO-DE-SOUZA1; GABRIELA PRISCILA DE SOUSA-MACIEL2; NIVIA DA SILVA DIAS-PINI3; MARIANNE GONÇALVES-BARBOSA4; POLIANA MARTINS-DUARTE5; PATRIK LUIZ PASTORI6

1 Student, Federal University of Ceará, Ceará, Brazil, [email protected], https://orcid.org/0000-0001-5524-6989. 2 Student, Federal University of Ceará, Ceará, Brazil, [email protected], https://orcid.org/0000-0001-8901-8749. 3 Biologist, Ph. D. in Entomology, researcher Brazilian Company of Agricultural Research (EMBRAPA), Ceará, Brazil, [email protected], https://orcid.org/0000-0002-3664-812X. 4 Agronomist, D. Sc. in Plant Science, Student Federal University of Ceará, Ceará, Brazil, [email protected], https://orcid.org/0000-0002-6395-1802. 5 Agronomist, M. Sc. in Plant Science, Student Federal University of Ceará, Ceará, Brazil, [email protected], https://orcid.org/0000-0002-3460-0456. 6 Agronomist, Ph. D. in Plant Science, Professor Federal University of Ceará, Ceará, Brazil, [email protected], https://orcid.org/0000-0003-1892-8029.

Abstract: The increased pest actions of the red cashew beetle Crimissa cruralis (Coleoptera: Corresponding author: Nivia da Silva Dias-Pini, Chrysomelidae in Brazilian populations of cashew Anacardium occidentale (Anarcardiaceae) Biologist, Ph. D. in Entomology, researcher is a cause for concern. The entomopathogenic fungus, Beauveria bassiana, was evaluated as Brazilian Company of Agricultural Research a biocontrol of larvae. The percent mortalities of second and third instar larvae produced by (EMBRAPA), Ceará, Brazil, nivia.dias@ in vivo application of a commercial formulation containing the fungal spores were evaluated. embrapa.br, https://orcid.org/0000-0002-3664- Treatment of larvae at concentrations of 1.2 x 106, 2.5 x 106, 3.7 x 107 e 5.0 x 107 conidia/ 812X. mL resulted in 49, 56, 66 and 72 % mortality within eight days, respectively. Based on the finding, it is recommended that the B. bassiana formulation should be employed at the lowest Suggested citation: concentrations (1.2 x 106 and 2.5 x 106 conidia/mL) with repeat application to avoid possible ARAÚJO-DE-SOUZA, S.; DE SOUSA- inactivation of the fungus by abiotic environmental factors. Cost-benefit assessments of the MACIEL, G. P.; DIAS-PINI, N. D. S.; use of this biological agent will be important for determining the economic feasibility of its GONÇALVES-BARBOSA, M.; MARTINS- application in the field. DUARTE, P.; PASTORI, P. L. 2019. Pathogenicity of Beauveria bassiana Keywords: Entomopathogenic fungus, controlled conditions, biological product, caju. (Deuteromycota: Hyphomycetes) to the red cashew beetle Crimissa cruralis (Coleoptera: Resumen: El aumento de la frecuencia de lo escarabajo rojo de la anacardiácea Crimissa Chrysomelidae: Galerucinae: Blepharida- cruralis (Coleoptera: Chrysomelidae) en poblaciones de Anacardium occidentale group) in laboratory conditions. Revista (Anarcardiaceae) del Brasil es motivo de preocupación. Se evaluó la mortalidad (en larvas Colombiana de Entomología 45 (2): e8606. de segundo y tercer instar) producida por la aplicación in vivo de una formulación comercial https://doi.org/10.25100/socolen.v45i2.8606 con esporas del hongo entomopatógeno, Beauveria bassiana. El tratamiento de larvas con concentraciones de 1,2 x 106, 2,5 x 106, 3,7 x 107 e 5,0 x 107 conidia/Ml resultó en 49, 56, Received: 16-Feb-2018 66 y 72 % de mortalidad dentro de ocho días, respectivamente. Se recomienda emplear la Accepted: 03-Aug-2019 formulación de B. bassiana en las concentraciones más bajas (1,2 x 106 y 2,5 x 106 conidia/ Published: 29-Jan-2020 mL) con aplicación repetida para evitar la posible inactivación de lo hongo por factores abióticos ambientales. Las evaluaciones de costo-beneficio del uso de este agente biológico Revista Colombiana de Entomología serán importantes para determinar la viabilidad económica de su aplicación en el campo. ISSN (Print): 0120-0488 ISSN (Online): 2665-4385 Palabras clave: Hongo entomopatógeno, condiciones controladas, producto biológico, cajú. http://revistacolombianaentomologia.univalle.edu.co/

Open access Introduction

BY-NC-SA 4.0. (https://creativecommons.org/licenses/by-nc-sa/4.0/deed.es) In 2017, the cultivated area of cashews, Anacardium occidentale Linnaeus (Anacardiaceae), in Brazil was 575,779 ha with a production of 99,073 tons of Publishers: Sociedad Colombiana de Entomología cashew nut extraction. The northeastern of Ceará, Rio Grande do Norte and Piaui SOCOLEN (Bogotá, D. C., Colombia) States together produced approximately 86,450 tons, which corresponds to 87.3 % http://www.socolen.org.co of the national production (Instituto Brasileiro de Geografia e Estatística 2017) of Universidad del Valle (Cali, Colombia) Brazil. http://www.univalle.edu.co/ The red cashew beetle, Crimissa cruralis Stal (Coleoptera: Chrysomelidae: © 2019 Sociedad Colombiana de Entomología Galerucinae: Blepharida-group), is considered to be a secondary pest in these - SOCOLEN y Universidad del Valle - Univalle cashew producing States and controlled naturally by the entomopathogenic fungus, Revista Colombiana de Entomología 2019, 45 (2): e8606 • Suyanne Araújo-de-Souza et al. 2/4

Beauveria bassiana (Balsamo) Vuillemin (Deuteromycota: 1A). After embryonic development, the larvae hatch and Hyphomycetes) (Mesquita and Braga Sobrinho 2014). move toward the plant canopy in search of leaves for feeding In recent years, the insect pest has often occurred in the (Fig. 1B). The larva starts feeding, causing a yield in the leaf municipality of Pacajus, State of Ceará. This is probably production. Throughout its development, the larvae’s feeding due to the low rainfall recorded which has been providing activity culminates with the defoliation of the plant (Fig. 1C). inadequate relative humidity conditions for the development In its last larval instar, the larva descends through the trunk to of the entomopathogenic fungus in the soil (Alves 1998), the soil, where it becomes a pupa. The pupa will remain in the thus resulting in a significant increase in the population of soil until the emergence of adults (Figs. 1D-1E). the insect. The entomopathogenic fungus, B. bassiana, can be In the field, C. cruralis mating occurs at any time of the employed as a biological control agent for management of day. Females oviposit in the cracks or holes in the trunk of immature and adult forms of C. cruralis (Mesquita et al. the plants. Shortly after oviposition, the females secrete a 2009); however, no information is available regarding the substance that covers the entire surface of the eggs that remain effects of the commercial B. bassiana-based products on in contact with the external environment outside the slit (Fig. C. cruralis. Therefore, the aim of the present study was to

Figure 1. A. Crimissa cruralis eggs in dwarf cashew trunk cracks. B. Larvae of the C. cruralis. C. Defoliation in dwarf cashew plants. D. Pre-pupae along the dwarf cashew trunk in the soil. E and F. Pupae in the soil and adult of the C. cruralis infected by Beauveria bassiana. Revista Colombiana de Entomología 2019, 45 (2): e8606 • Suyanne Araújo-de-Souza et al. 3/4 evaluate the outcomes of in vivo applications of a commercial Table 1. Percentage confirmed mortality of second and third instar formulation of B. bassiana on larvae of C. cruralis. larvae of C. cruralis (Coleoptera: Chrysomelidae) caused by different concentrations of B. bassiana- based product (Ballvéria®).

Materials and methods Concentrations Mortality (%) (Con./mL) Total Confirmed Corrected Larvae of C. cruralis were collected from cashew trees cultivated in the experimental field of “Embrapa Agroindústria Control 18.0 ± 8.0 a 0.0 ± 0.0 a - Tropical” (Pacajus, CE, Brazil: 4°10’35ˮS, 38°28’19ˮW; 1.2 x 106 55.0 ± 8.0 b 49.0 ± 8.0 b 37.6 ± 13.0 a altitude 79 m). Second and third instar larvae were separated 2.5 x 106 56.2 ± 7.0 b 56.2 ± 7.0 b 47.3 ± 11.0 a based on their size (1 cm) and placed individually into glass 3.7 x 107 66.0 ± 11.0 b 66.0 ± 11.0 b 60.3 ± 13.0 a tubes (diameter 8.5 x 2.5 cm) containing new cashew leaves. 7 The rearing conditions for the larvae were 26 ± 2 °C, 50 ± 10 5.0 x 10 81.0 ± 9.0 c 72.0 ± 10.0 c 69.3 ± 9.0 a % relative humidity under a 12 h photophase and leaf material P-value 0.009 0.009 0.46 was replaced daily. Means followed by the same letter, in the column, do not differ among themselves by the The commercial formulation Ballvéria® (Ballagro Agro Kruskal-Wallis test, at 5 % probability. Tecnologia, Bom Jesus dos Perdões, SP, Brazil) employed in the experiment is a wettable powder containing spores of B. bassiana strain IBCB66. Powdered bioinsecticide was diluted when populations of this pest reach higher levels. We suggest in distilled water (0.02, 0.1, 1.0 and 2.0 g/L) and a 1.0 mL that the successful control of C. cruralis infestation on cashew dose was sprayed over 5 larvae with the help of a micro-paint trees could be carried out using the commercial formulation of air compressor Wimpel ® (15-25 PSI). Pure distilled water B. bassiana at the lowest concentration tested (i.e. 1.2 x 106, was used as the negative control. Following application of 2.5 x 106 conidia/mL) but with repeated applications. Thus, the treatments, larvae were transferred to Petri dishes (5 cm when the biocontrol is introduced into the field and the pest diameter) and maintained at 26 ± 2 °C and 50 ± 10 % relative comes into contact with the fungus, even in low concentration, humidity in a biochemical oxygen demand incubator. there is a possibility of causing insect mortality. This strategy Mortality was defined as the total immobilization of is based on the acceptable mortality rate (49 to 56 %) induced larvae and observations were carried out every 24 h over 8 by the two concentrations of insecticide and the necessity to consecutive days. In order to confirm the death was caused prevent inactivation of the agent by abiotic factors such as by B. bassiana, dead larvae were immersed in 70 % ethanol solar and ultraviolet radiation. Although a single application for 60 s to remove external contaminants and placed in sterile of insecticide at a high concentration could produce greater Petri dishes (5 cm diameter) lined with damp filter paper mortality, it is possible that the fungal spores would not and closed with plastic film for eight days to allow fungal survive exposure to the environmental conditions and on- sporulation. Thus, the percentage of dead larvae in each going control of the pest would most likely fail (Oliveira et al. treatment that presented B. bassiana spores in their tegument 2016). In this context, it is worth noting that the manufacturer was considered to be the confirmed mortality rate. of the insecticide recommends application of the product at The experimental design was fully randomized and 1.25 g /L p.c. (1.2 x 107 conidia/mL) for soybean plantations. consisted of five treatments and five replicate dishes each At the maximum concentration, the high mortality with five larvae/Petri dish. The confirmed percent mortality rate for the red cashew beetle larvae (72 %) by the B. caused by each of the bioinsecticide concentrations was bassiana formulation in this study was close to when the corrected using Abbott’s formula (Abbott 1925), and the entomopathogenic fungus, Ophiocordyceps melolonthae significant differences amongst treatments were determined (Ascomycota: Hypocreales: Ophiocordycipitaceae) was using Kruskal-Wallis test (α = 0.05). Statistical analyses were applied against larvae of the white grub Dyscinetus gagates carried out using PAST software (Hammer et al. 2001). Burmeister (75 %) (Coleoptera: Melolonthidae) (Salgado- Neto et al. 2017). In addition, tests per formed by Rezende et Results and discussion al. (2009) revealed that suspensions of B. bassiana containing 107 conidia/mL induced 95 and 62.5 % mortality, respectively, Treatment of larvae with the commercial formulation of B. against larvae and adults of the litter beetle Alphitobius bassiana at the lowest concentration tested (1.2 x 106 conidia/ diaperinus (Panzer, 1797) (Coleoptera: Tenebrionidae). These mL) resulted in 49 % mortality of C. cruralis over eight days. findings suggest that larvae are more susceptible to various The confirmed mortality percentage increased progressively strains of B. bassiana in comparison with adult insects. with greater concentration of the bioinsecticide and attained The present study has confirmed the efficacy of the B. 56, 66 and 72 %, respectively, at 2.5 x 106, 3.7 x 107 e 5.0 bassiana product against C. cruralis larvae in laboratory x 107 conidia/mL, giving an overall increase in mortality conditions. However, alongside the health and environmental of 1.46-fold between the lowest and highest concentrations benefits that normally accrue from the use of biological employed (Table 1). agents in the control of crop pests, the strategy must be Associated with the main pests of the cashew tree there is economically feasible. Based on our suggested protocol a rich fauna of beneficial insects (predators and parasitoids) involving three treatments with B. bassiana product, each and entomopathogenic fungi. Among the entomopathogenic at a concentration of 2.5 x 106 conidia/mL and applied at a mitosporic fungi is B. bassiana which naturally infects C. rate of 200 L/ha, we calculate that the cost of treating 1 ha cruralis (Mesquita and Braga Sobrinho 2014). of cashew orchard would be R$ 42.00 (~ 13.2 USD) given Despite the natural occurrence of this fungus, the that the cost of the product is currently R$ 140.00 per 200 g. introduction of commercial formulation of B. bassiana in the Although it appears that our recommended procedure would field would be interesting for achieving an effective control be advantageous economically, the exact cost of the treatment Revista Colombiana de Entomología 2019, 45 (2): e8606 • Suyanne Araújo-de-Souza et al. 4/4 will vary depending on the density of trees in the plantation HAMMER, Ø.; HARPER, D. A. T.; RYAN, P. D. 2001. PAST: and the number of applications determined to be necessary. Paleontological statistics software package for education and data analysis. Palaeontologia Electronica 4 (1), art. 4. Available Conclusions in: http://palaeo-electronica.org/2001_1/past/past.pdf [Review date: 16 September 2019] INSTITUTO BRASILEIRO DE GEOGRAFIA E ESTATÍSTICA. Field studies involving cost-benefit evaluations will be 2017. Castanha-de caju. Available in: https://sidra.ibge. required to enable cashew producers to determine whether gov.br/home/lspa/brasil and https://sidra.ibge.gov.br/ the use of B. bassiana is economically feasible. Moreover, tabela/1618#resultado [Review date: 16 September 2019]. it will be important to convey comprehensive information MESQUITA, A. L. M.; BRAGA SOBRINHO, R. 2014. Ocorrência about the formulation (what the product is, how it works e importância de inimigos naturais de pragas em cajueiro no and how it should be used) to farmers so that they can estado do Ceará (Brasil). Essentia 16 (1): 103-110. make knowledgeable decisions regarding pest control of MESQUITA, A. L. M.; FANCELLI, M.; BRAGA SOBRINHO, this chrysomelid pest. It is important to emphasize that the R. 2009. Ocorrência e importância de inimigos naturais de production of “caju” (fruit of the cashew tree) comprises pragas em cultivo de cajueiro orgânico. Embrapa Agroindústria mixed systems, i.e. it ranges from high-tech farms that grow Tropical-Comunicado Técnico, 4 p. OLIVEIRA, M. T.; MONTEIRO, A. C.; LA SCALA-JÚNIOR, N.; genetically improved varieties to extractive farms that exploit BARBOSA, J. C.; MOCHI, D. A. 2016. Sensibilidade de isolados natural resources. Hence, the economic viability of using the de fungos entomopatogênicos às radiações solar, ultravioleta e à B. bassiana product must be evaluated for each particular temperatura. Arquivos do Instituto biológico 83: 1-7. https://doi. production system alongside all other biocontrol measures org/10.1590/1808-1657000042014 being used on the array of pathogens of cashew in this part of REZENDE, S. R. F.; CURVELLO, F. A.; FRAGA, M. E.; REIS, Brazil. Furthermore, application of the B. bassiana product in R. C. S.; CASTILHO, A. M. C.; AGOSTINHO, T. S. P. 2009. the field must be validated since cashew cultures are grown Control of the Alphitobius diaperinus (Panzer) (Coleoptera: mainly in semi-arid regions that receive little precipitation Tenebrionidae) with entomopathogenic fungi. Revista Brasileira and the effect of such conditions on the fungi are currently de Ciência Avícola 11 (2): 121-127. https://doi.org/10.1590/ unknown. Thus, these two issues require further evaluation S1516-635X2009000200008 SALGADO-NETO, G.; VAZ, M. B.; GUEDES, J. V. C. 2017. and we commend them for future research based on the White grub (Coleoptera: Melolonthidae) mortality induced by promising results reported herein. Ophiocordyceps melolonthae. Ciência Rural 47 (6): e20160788. https://doi.org/10.1590/0103-8478cr20160788 Acknowledgements ______This work was supported by the National Council for Origen and funding Scientific and Technological development (CNPq) and the Embrapa Agroindústria Tropical funding this research. Brazilian Company of Agricultural Research (EMBRAPA). Author contribution Literature cited Suyanne Araújo-de-Souza, Gabriela Priscila de Sousa-Maciel and ABBOTT, W. S. 1925. A method for computing the effectiveness of Poliana Martins-Duarte: experiments and article writing. Marianne insecticides. Journal of Economic Entomology 18 (15): 265-267. Gonçalves-Barbosa: data analysis and article writing. Nívia da Silva https://doi.org/10.1093/jee/18.2.265a Dias-Pini and Patrick Luiz Pastori: research planning, manuscript ALVES, S. B. 1998. Controle microbiano de insetos. São Paulo: corrections and advise to the first author. Manole, 386 p.